Compositions containing metal nanoparticles, methods for making same, and uses thereof

Abstract

A composition comprising a plurality of particle clusters in a carrier, at least one of the particle clusters comprising a plurality of metal nanoparticles, and the composition of the at least one particle cluster is configured such that the composition has an absorbance spectrum peak above about 900 nm. A method of making the composition includes reacting a metal nanoparticle precursor with a stabilizing agent to produce functionalized metal nanoparticles, dispersing the functionalized metal particles in a clustering agent to form particle clusters, and resuspending the particle clusters in a carrier to form the composition. Uses of the composition include as a contrast agent for imaging.

Description

Detailed Description of the Invention

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 300,732, filed January 19, 2022, the entire contents of which are incorporated herein by reference.

[0002] [Field] The present disclosure relates to compositions including metal nanoparticles, methods for making same, and uses thereof.

[0003] [background] Nanoparticles are used in many applications including, for example, catalysis, energetic materials / photonics, and imaging as contrast agents. Varying the base material, reaction conditions, and surface chemistry of the nanoparticles can alter their physical, chemical, and optical properties, resulting in highly versatile contrast agents with a very wide variety of applications.

[0004] One such use is as a contrast agent in biomedical imaging using the second near-infrared (NIR-II) window around 1000 to 1700 nm, where photon penetration in vivo is maximal due to minimized absorption and scattering by blood and tissue, allowing high signal-to-noise images of deep tissues to be obtained.

[0005] With regard to the medical field, spherical gold nanoparticles (AuNPs) are used in a variety of different imaging modalities, such as OCT, due to their well-established biocompatibility, physicochemical and plasmonic tunability, and targetability. One of the main drawbacks of conventional AuNPs, whether spherical or of another shape, is that their localized surface plasmon resonance wavelength (500 nm-650 nm) lies within the visible light spectrum, meaning that they are incompatible with more commonly used imaging modalities that exploit the NIR-II window (>1000 nm) of biological imaging.

[0006] More recently, gold-based compositions that resonate above 1000 nm have been proposed for biomedical applications, but suffer from several drawbacks, such as lack of ease of preparation, scalability, and signal strength.

[0007] There are other metallic plasmonic materials that can reach these NIR-II wavelengths, such as copper sulfide and silver, but gold remains the standard due to its chemical inertness and well-documented biological clearance.

[0008] Thus, there is a need for improved compositions comprising nanoparticles.

[0009] [overview] It is an object of the present invention to alleviate at least some of the shortcomings present in the prior art.Embodiments of the present technology have been developed based on the inventors' realization that there is a need for improved compositions comprising nanoparticles for imaging purposes.

[0010] The inventors have found that the light absorption properties of a composition comprising metal nanoparticles can be adjusted by providing clusters of metal nanoparticles and adapting the properties of the clusters to adapt the optical properties of the composition.The inventors have noted that adapting the properties of the clusters, such as one or more of the number of metal nanoparticles in each cluster, the diameter of each cluster, the shape of each cluster, the loading of metal nanoparticles in each cluster, and the size distribution of the clusters in the carrier, can adapt the light absorption of the composition.In certain embodiments, the composition may have optical properties that shift to a second near infrared (NIR-II) window.

[0011] Without wishing to be bound by any theory, the inventors hypothesize that such clusters of metal nanoparticles may undergo extensive plasmonic hybridization, a phenomenon that occurs when two or more plasmonic fields are brought into close proximity, creating a red-shift in the overall plasmonic absorption band of the material due to destructive interference.

[0012] Furthermore, the inventors have developed compositions comprising such clusters that are scalable, water soluble and uniformly distributed, and therefore may be suitable for biomedical applications.

[0013] This differs from prior art reports of nanoparticle clusters composed of less than 10 particles in close proximity and with plasmon red shifts of less than 100 nm. (Liu X et al., ChemPhysChem, 2007;8(6):906-912; Nguyen VP et al., Nat Commun, 2021;12(1):34; Norman TJ et al., J Phys Chem B, 2002;106(28):7005-7012; Kim J et al., Nano Converg, 2020;7(1):5).

[0014] Turek et al. (Turek VA et al., ACS Photonics, 2016;3(1):35-42) used microemulsion techniques to form gold superclusters, but this clustering occurred only as a shell to an emulsion core, resulting in optical properties similar to those of standard gold nanoparticles.

[0015] Kwon et al. (Kwon N et al., Nano Lett, 2018;18(9):5927-5932) also demonstrated the solvophobic formation of gold superclusters using oleylamine-capped gold nanoparticles prepared in a flask in a hot oil bath that aggregated when added to ethanol but dispersed in hexane or other organic solvents. Although these clusters exhibited absorbance in the NIR, they were highly heterogeneous in terms of size and shape distribution, were not water-dispersible, and had no clearly defined NIR absorbance peaks (likely due to size and shape heterogeneity in the solution).

[0016] Additionally, the inventors have developed methods for tailoring the size and shape of nanoparticle clusters using stabilizing agents and pre-capping solvents. Certain embodiments of such methods exploit the solvophobic effect as a driving force. Embodiments of such methods include "locking" the particle cluster morphology to ensure a uniform suspension of the particle clusters in the carrier and the ability to transfer the particle clusters into an aqueous solvent without loss of morphology.

[0017] From one aspect, there is provided a composition comprising a plurality of particle clusters in a carrier, at least one particle cluster comprising a plurality of metal nanoparticles, and the configuration of the at least one particle cluster is such that the composition has an absorbance spectral peak greater than about 900 nm.

[0018] In certain embodiments, metal nanoparticles refer to particles having metallic properties, such as, but not limited to, metals, metal alloys, or metal oxides. Nanoparticles refer to particles having a size range of about 1 nm to about 500 nm. In some embodiments, nanoparticles have a size range of about 1 nm to about 100 nm.

[0019] In certain embodiments, the metal nanoparticles include silver or gold particles.

[0020] In certain embodiments, at least one of the particle clusters comprises a coating layer. The coating layer comprises a polymer that can be amphiphilic.

[0021] In certain embodiments, the plurality of particle clusters has a substantially uniform size distribution, which in certain embodiments means that the composition has a polydispersity index of 0.3 or less as measured by transmission electron microscopy.

[0022] In certain embodiments, at least one particle cluster is water-dispersible.

[0023] In certain embodiments, the carrier is an aqueous solution. The carrier may be saline, water, or 5% dextrose in water. Such compositions may be used for biomedical applications.

[0024] In certain other embodiments, the carrier is a polar organic solvent.Such compositions may be used in non-biomedical applications.

[0025] In certain embodiments, each metal nanoparticle in at least one particle cluster is functionalized with a stabilizing agent. The stabilizing agent may include one or more of an amine, thiol, or carboxylic acid head group and a hydrophobic tail of any length and saturation. Optionally, the stabilizing agent is oleylamine, octadecene thiol, oleic acid, or a combination thereof.

[0026] In certain embodiments, the configuration of the at least one particle cluster includes one or more of a given number of metal nanoparticles in the at least one particle cluster, a size of the at least one particle cluster, a shape of the at least one particle cluster, and a given packing of metal particles in the at least one particle cluster, hi certain embodiments, the at least one particle cluster includes at least three layers of nanoparticles in the xyz plane.

[0027] In certain embodiments, the absorbance spectrum of the composition is between about 900 nm and about 1700 nm, between about 900 nm and about 1600 nm, between about 900 nm and about 1500 nm, between about 900 nm and about 1400 nm, between about 900 nm and about 1300 nm, between about 950 nm and about 1700 nm, between about 950 nm and about 1600 nm, between about 950 nm and about 1500 nm, between about 950 nm and about 1400 nm, or between about 950 nm and about 1300 nm, between about 1000 nm and about 1700 nm, between about 1000 nm and about 1600 nm, between about 1000 nm and about 1500 nm, between about 1000 nm and about 1400 nm, or between about 1000 nm and about 1300 nm.

[0028] In certain embodiments, at least one particle cluster is substantially spherical.

[0029] In certain embodiments, the polymer comprises one or more of polyethylene glycol, polyvinyl chloride, poly-l-lysine, polylactic acid, poly(lactic-co-glycolic acid), polystyrene, and polyvinylpyrrolidone, and / or block copolymers derived therefrom. In certain embodiments, the block copolymer is derived from polyethylene glycol. In certain embodiments, the block copolymer comprises polyoxyalkylenes with saturated or unsaturated alkyl chains (e.g., BRIJ™ family); polyoxyethylene derivatives of saturated or unsaturated fatty acids, and / or high molecular weight polyoxyalkylene ethers with water-soluble, surface active wetting properties (e.g., MYRJ™ family).

[0030] In certain embodiments, the coating layer comprises multiple coating layers on the at least one particle cluster. In certain embodiments, where the coating layer comprises an amphiphilic polymer, multiple amphiphilic coating layers are provided.

[0031] In certain embodiments, the composition further comprises a targeting agent attached to the surface of the particle cluster. In certain embodiments where a coating is on the particle cluster, the targeting agent is attached to the coating (e.g., an amphiphilic polymer).

[0032] In certain embodiments, the targeting agent comprises one or more of a small molecule ligand, a peptide, a polymer, a nucleic acid construct (including DNA and RNA aptamers), a protein, a nanobody, an affibody, a minibody, a diabody, or an antibody.

[0033] In certain embodiments, the targeting agent binds to a marker of intravascular inflammation.

[0034] In certain embodiments, the targeting agent specifically binds to one or more of P-selectin, E-selectin, and VE-cadherin, hi some such embodiments, the targeting agent is a ligand for P-selectin, E-selectin, or VE-cadherin.

[0035] In certain embodiments, the targeting agent comprises a mixture of polymers, the mixture comprising a mixture ratio of fucose:sulfate (e.g., 1:2), galactose:sulfate (e.g., 1:2), or fucose:galactose:sulfate (e.g., 1:1:1).

[0036] In certain embodiments, the average diameter of the particle clusters is from about 250 nm to about 1500 nm, or from about 300 nm to about 500 nm, or about 419 nm.

[0037] In certain embodiments, the metal nanoparticles are substantially spherical.

[0038] In certain embodiments, the average particle size of the metal nanoparticles ranges from about 2 nm to about 50 nm, hi certain embodiments, the metal nanoparticles have an average particle size of about 9 nm.

[0039] From another aspect, a composition is provided that includes a plurality of particle clusters in a carrier, at least one particle cluster of the plurality of particle clusters includes a plurality of metal nanoparticles, each metal nanoparticle is functionalized with a stabilizing agent, and at least one particle cluster has a coating layer. In certain embodiments, the coating layer is a polymer, such as an amphiphilic polymer.

[0040] In certain embodiments, the configuration of the metal particles within the at least one particle cluster is configured such that the composition has an absorbance spectral peak above about 900 nm.

[0041] In certain embodiments, the plurality of particle clusters has a substantially uniform size distribution, which in certain embodiments means that the composition has a polydispersity index of 0.3 or less as measured by transmission electron microscopy.

[0042] In certain embodiments, at least one particle cluster is water dispersible. In certain embodiments, the carrier is an aqueous solution. In certain embodiments, the carrier is saline, water, or 5% dextrose in water.

[0043] In other embodiments, the carrier is a polar organic solvent.

[0044] In certain embodiments, the stabilizing agent comprises one or more of an amine, a thiol, a carboxylic acid head group, and a hydrophobic tail.

[0045] In certain embodiments, the configuration of nanoparticles in the particle clusters includes a given number of metal particles in at least one particle cluster and / or a given loading of metal particles in at least one particle cluster, hi certain embodiments, at least one particle cluster includes at least three layers of nanoparticles in the xyz plane.

[0046] In certain embodiments, the absorbance spectrum of the composition is between about 900 nm and about 1700 nm, between about 900 nm and about 1600 nm, between about 900 nm and about 1500 nm, between about 900 nm and about 1400 nm, between about 900 nm and about 1300 nm, between about 950 nm and about 1700 nm, between about 950 nm and about 1600 nm, between about 950 nm and about 1500 nm, between about 950 nm and about 1400 nm, or between about 950 nm and about 1300 nm, between about 1000 nm and about 1700 nm, between about 1000 nm and about 1600 nm, between about 1000 nm and about 1500 nm, between about 1000 nm and about 1400 nm, or between about 1000 nm and about 1300 nm.

[0047] In certain embodiments, at least one particle cluster is substantially spherical.

[0048] In certain embodiments, the amphiphilic polymer comprises one or more of polyethylene glycol, polyvinyl chloride, poly-l-lysine, polylactic acid, PLGA, polystyrene, polyvinylpyrrolidone, and / or block copolymers derived therefrom. In certain embodiments, the block copolymer is derived from polyethylene glycol, including but not limited to polyoxyalkylenes with saturated or unsaturated alkyl chains (e.g., BRIJ™ family); polyoxyethylene derivatives of saturated or unsaturated fatty acids, and / or high molecular weight polyoxyalkylene ethers with water-soluble, surface active wetting properties (e.g., MYRJ™ family).

[0049] In certain embodiments, the metal nanoparticles include particles that are generally metallic and may include, for example, a metal, a metal alloy, or a metal oxide, hi certain embodiments, the metal nanoparticles include silver or gold particles.

[0050] In certain embodiments, multiple coating layers are provided on the particle clusters.

[0051] In certain embodiments, the composition further comprises a targeting agent attached to the surface or coating of the particle cluster (eg, an amphiphilic polymer).

[0052] In certain embodiments, the targeting agent comprises one or more of a small molecule ligand, a peptide, a polymer, a nucleic acid construct (including DNA and RNA aptamers), a protein, a nanobody, an affibody, a minibody, a diabody, or an antibody.

[0053] In certain embodiments, the targeting agent binds to a marker of intravascular inflammation.

[0054] In certain embodiments, the targeting agent specifically binds to one or more of P-selectin, E-selectin, and VE-cadherin, hi some such embodiments, the targeting agent is a ligand for P-selectin, E-selectin, or VE-cadherin.

[0055] In certain embodiments, the targeting agent comprises a mixture of polymers, the mixture comprising a mixture ratio of fucose:sulfate (e.g., 1:2), galactose:sulfate (e.g., 1:2), or fucose:galactose:sulfate (e.g., 1:1:1).

[0056] In certain embodiments, the average diameter of the particle clusters is from about 250 nm to about 1500 nm, or from about 300 nm to about 500 nm.

[0057] In certain embodiments, the metal nanoparticles are substantially spherical.

[0058] In certain embodiments, the average particle size of the metal nanoparticles ranges from about 2 to about 50 nm, hi certain embodiments, the metal nanoparticles have an average particle size of about 9 nm.

[0059] In certain embodiments, the composition is suitable for use as an imaging agent.

[0060] From yet another aspect, there is provided a method of making an embodiment of the composition as claimed and described herein, the method comprising (i) reacting a metal nanoparticle precursor with a stabilizing agent to produce functionalized metal nanoparticles, (ii) dispersing the functionalized metal particles in a clustering agent to form metal particle clusters, and (iii) resuspending the metal particle clusters in a carrier to form the composition.

[0061] In certain embodiments, the metal nanoparticles are generally metallic and may include metals, metal alloys, or metal oxides.

[0062] The metal may comprise silver or gold, and the metal nanoparticle precursor may comprise a gold particle precursor or a silver particle precursor, respectively. In certain embodiments, the metal particle precursor is HAuCl4 or AgNO3.

[0063] In certain embodiments, the stabilizer comprises one or more of an amine, thiol, or carboxylic acid head group and a hydrophobic tail of any length and degree of saturation, optionally the stabilizer is oleylamine, octadecene thiol, oleic acid, or a combination thereof.

[0064] In certain embodiments, the clustering agent is an organic solvent. The clustering agent may be one or more of butanol, ethanol, petroleum ether, butanol-hexane. In certain embodiments, the clustering agent may comprise a modified polymer or block copolymer that includes hydrophobic and hydrophilic domains, such as, but not limited to, Pluronic™ family members, such as F127, MYRJ™, and / or BRIJ™ family members, such as polyethylene oxide (40) stearate and polyvinylpyrrolidone.

[0065] In certain embodiments, the reaction includes heating the metal nanoparticle precursor with a stabilizing agent. The heating may include microwave heating. In certain other embodiments, the heating includes one or more of oven heating, oil bath heating, water bath heating, or mantle heating. In certain embodiments, microwave heating can significantly reduce the reaction time for cluster formation.

[0066] In certain embodiments, the carrier in the composition is an aqueous solution, and the method further comprises separating the particle clusters from the clustering agent and suspending them in the aqueous solution.

[0067] In certain embodiments, separation is by centrifugation or by sedimentation, while in other embodiments, separation is by size exclusion chromatography or magnetic separation.

[0068] In certain embodiments, the method further includes coating the metal particle clusters in a coating layer.

[0069] From another aspect, there is provided a method of biomedical imaging comprising administering an imaging agent to a subject and imaging the imaging agent in the subject, the imaging agent comprising a composition described and / or claimed herein. In certain embodiments, the biomedical imaging comprises optical coherence tomography (OCT). The OCT may comprise intravascular OCT.

[0070] In one particular embodiment, imaging relies on NIR light at wavelengths from about 1000 nm to about 1700 nm.

[0071] In certain other embodiments, imaging relies on one or more of: (i) absorption of x-rays; (ii) diffraction of x-rays; (iii) absorption of light; and (iv) detection by an ultrasonic transducer.

[0072] From yet another aspect, there is provided a composition as described and / or claimed herein for use in imaging, such as biomedical imaging.

[0073] From yet another aspect, there is provided a composition as described and / or claimed herein for use as an imaging agent, which may be used during imaging using modalities such as OCT, x-ray, CT, synchrotron, and photoacoustic.

[0074] From another aspect, there is provided an imaging agent for biomedical imaging comprising a composition as described and / or claimed herein.

[0075] For a better understanding of the present invention, and to show more clearly how the same may be carried into effect, reference will now be made by way of example to the accompanying drawings which illustrate aspects and features in accordance with embodiments of the invention. [Brief description of the drawings]

[0076] [Figure 1] 1 is a flow diagram of a method for making a composition with metal nanoparticles, according to certain embodiments of the present technology. [Diagram 2] FIG. 1 is a schematic diagram of a method for making a composition with metal nanoparticles, according to one particular embodiment of the present technology. [Diagram 3] FIG. 2 illustrates the morphology and size distribution of particle clusters of metal nanoparticles in a composition, according to certain embodiments of the present technology. [Figure 4] 1 is a graph showing the aqueous size and dispersibility of particle clusters of metal nanoparticles in a composition in accordance with certain embodiments of the present technology. [Diagram 5] 1 is a graph showing absorbance spectra of particle clusters of metal nanoparticles with and without a coating in a composition in accordance with certain embodiments of the present technology; [Figure 6] FIG. 2 illustrates finite-difference time-domain simulation data of particle clusters of metal nanoparticles in a composition, in accordance with certain embodiments of the present technique. [Figure 7] 1 is a graph showing finite-difference time-domain simulation data of particle clusters of metal nanoparticles in a composition using a fixed unit cell, in accordance with certain embodiments of the present technology; [Figure 8] 1 is a graph showing finite-difference time-domain simulation data of particle clusters of metal nanoparticles in compositions using various unit cells, in accordance with certain embodiments of the present technology; [Figure 9] 1 is a graph showing finite-difference time-domain simulation data of particle clusters of metal nanoparticles in compositions using various coating layer thicknesses in accordance with certain embodiments of the present technology; [Figure 10] FIG. 1 shows a transmission electron microscope image and absorbance spectrum of a composition including metal nanoparticle clusters in saline, in accordance with certain embodiments of the present technology. [Figure 11]FIG. 1 shows intravascular optical coherence tomography images of a composition comprising metal nanoparticle clusters in a carrier compared to reference gold nanoparticles, in accordance with certain embodiments of the present technology. [Figure 12] FIG. 1 shows intravascular optical coherence tomography contrast enhancement between a composition comprising metal nanoparticle clusters in a carrier compared to reference gold nanoparticles, in accordance with certain embodiments of the present technology. [Figure 13] FIG. 13 shows intravascular optical coherence tomography pullback images in a vascular phantom between compositions comprising metal nanoparticle clusters in a carrier compared to reference gold nanoparticles, in accordance with certain embodiments of the present technology. [Figure 14] FIG. 1 shows intravascular optical coherence tomography of a Sprague-Dawley rat abdominal aorta that was flushed sequentially with saline, with a composition comprising metal nanoparticle clusters in a carrier, and again with saline, in accordance with certain embodiments of the present technology. [Figure 15A] FIG. 1 illustrates a method for functionalizing the surface polymer of gold particle clusters (AuSCs) with targeting ligands (1-3) according to certain embodiments of the present technology. [Figure 15B] 1 shows transmission electron micrographs of AuSCs functionalized with different combinations of targeting ligands as shown (left panel), according to certain embodiments of the present technology. The extent of AuSC binding to P-selectin in vitro for different formulations of AuSC targets is shown in the right panel. *p<0.05, **p<0.01, ****p<0.001 [Figure 15C] Photographs showing intravascular optical coherence tomography of Sprague-Dawley rat abdominal aorta after induction of intra-arterial inflammation, before and after introduction of non-targeted (left) or targeted AuSCs (middle and right). According to certain embodiments of the present technology, the artery was sequentially flushed with saline, with a composition comprising metal nanoparticle clusters in a carrier, and again with saline. [Figure 15D]FIG. 2 shows a 400 MHz 1H NMR spectrum of as-synthesized FucoPEO prior to particle conjugation, according to certain embodiments of the present technology. [Figure 15E] FIG. 2 shows a 400 MHz 1H NMR spectrum of as-synthesized GalaPEO prior to particle conjugation, according to certain embodiments of the present technology. [Figure 15F] FIG. 2 shows a 400 MHz 1H NMR spectrum of as-synthesized SulfoPEO prior to particle conjugation, according to certain embodiments of the present technology. [Figure 15G] 1 shows a MALDI-TOF spectrum for FucoPEO, according to certain embodiments of the present technology. The spectrum shows a central mass of M+Na, with other peaks being different ethylene oxide chain lengths with the same functional group. [Figure 15H] 1 shows a MALDI-TOF spectrum for GalaPEO, according to certain embodiments of the present technology. The spectrum shows a central mass of M+Na, with other peaks being different ethylene oxide chain lengths with the same functional group. [Figure 15I] 1 shows a MALDI-TOF spectrum for SulfoPEO, according to certain embodiments of the present technology. The spectrum shows a central mass of M+Na, with other peaks being different ethylene oxide chain lengths with the same functional group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] [Detailed Description] In order to provide a clear and consistent understanding of the terms used herein, certain definitions are provided below. Furthermore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the inventor pertains.

[0078] The use of the word "a" or "an," when used in conjunction with the term "comprising" in the claims and / or specification, may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one." Similarly, the word "another" may mean at least a second or more.

[0079] As used in the specification and claim(s), the words "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "include" and "includes"), or "containing" (and any form of containing, e.g., "contain" and "contains") are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0080] The term "about" is used to indicate that a value includes an inherent variation of error for the device or method being employed to determine the value.

[0081] The terms "derivative" and "variant" are used interchangeably herein.

[0082] An embodiment of the present technology includes a composition having a cluster of metal nanoparticles in a carrier. The optical properties, such as the absorption spectrum, of the composition can be tailored for a given use by adapting one or more cluster parameters, such as the cluster diameter, the number of nanoparticles in the cluster, the loading of the metal nanoparticles in the cluster, and the size or shape distribution of the cluster in the carrier. An embodiment of the present technology includes a method of making such a composition.

[0083] composition In certain embodiments, the composition comprises particle clusters comprising metal nanoparticles in a support.

[0084] Metal Nanoparticles and Particle Clusters The metal nanoparticles may include any suitable metal particles, such as metal alloys, metal oxides, and pure metals. Example metals include, but are not limited to, gold, silver, copper, palladium, and manganese oxides. The precursor to the gold particles may include HAuCl4.

[0085] In certain embodiments, the metal particles have diameters within the range of about 1 to 100 nm, about 1 to about 90 nm, about 1 to about 80 nm, about 1 nm to about 70 nm, about 1 nm to about 60 nm, about 1 nm to about 50 nm, or about 2 nm to about 50 nm. In certain embodiments, the metal nanoparticles include gold nanoparticles having a diameter of about 5-15 nm, or about 9 nm.

[0086] The size distribution of the metal nanoparticles within the cluster may be substantially uniform. For example, the diameter of the metal nanoparticles may range from about 8 nm to about 11 nm, with a median and average diameter of 9 nm. In other embodiments, the size distribution of the metal nanoparticles within the cluster may be substantially non-uniform. For example, the metal nanoparticles may have a diameter between about 1 nm and about 100 nm.

[0087] The particle clusters in the composition are substantially spherical in certain embodiments.

[0088] In certain embodiments, the size of at least some of the particle clusters ranges from about 250 nm to about 1500 nm, from about 300 nm to about 1400 nm, from about 300 nm to about 1300 nm, from about 300 nm to about 1200 nm, from about 300 nm to about 1100 nm, from about 300 nm to about 1000 nm, from about 300 nm to about 900 nm, from about 300 nm to about 800 nm, from about 400 nm to about 800 nm, from about 500 nm to about 800 nm, from about 300 nm to about 700 nm, from about 300 nm to about 600 nm, from about 300 nm to about 500 nm, from about 400 nm to about 600 nm, or from about 400 nm to about 500 nm.

[0089] The size of the particle clusters may be measured by any known method, such as image analysis of electron microscopy images of the particle clusters, or using a particle sizer of the particle clusters in solution.

[0090] The particle clusters are, in certain embodiments, substantially uniformly sized, which in certain embodiments means that the composition has a polydispersity index of 0.3 or less as measured by transmission electron microscopy.

[0091] The packing of the metal nanoparticles can be defined as the interparticle distance, which in certain embodiments is defined as the unit cell volume where the corner-to-corner distance of the unit cell represents the interparticle distance between two metal nanoparticles.

[0092] For particle clusters containing gold nanoparticles, the unit cell can be assumed to be a face-centered cubic unit cell (i.e., the unit cell lengths in the x, y, and z directions are equal). The unit cell volume, and therefore the packing of gold nanoparticles within the particle cluster, can be determined from the measured sizes of the particle cluster and gold nanoparticles. The edge length of the unit cell can be calculated to be 2.828 x the atomic radius of gold(144), which equals 0.4073 nm.

[0093] In certain embodiments, the packing of the metal nanoparticles is uniform throughout the supercluster. In other embodiments, the packing of the metal particles varies from the inner portion to the outer portion of the particle cluster. For example, the metal nanoparticles may be more densely packed in the inner portion of the particle cluster compared to the outer portion of the particle cluster.

[0094] In one particular embodiment, the metal nanoparticle packing is greater than 13 nm in unit cell volume. 3 , 14nm 3 , or 15 nm 3 For example, the filling of the inner part of the particle cluster is 14 nm 3 and the packing of the outer portion of the particle cluster may comprise a unit cell volume of 15 nm 3 In another example, the packing of metal particles extending radially from the inner portion to the outer portion may include 13 nm 3 , 14nm 3 , and 15 nm 3 The volume of the unit cell may vary gradually, for example having a unit cell volume of

[0095] The absorbance spectrum of the composition is between about 900 nm and about 1700 nm, between about 900 nm and about 1600 nm, between about 900 nm and about 1500 nm, between about 900 nm and about 1400 nm, between about 900 nm and about 1300 nm, between about 950 nm and about 1700 nm, between about 950 nm and about 1600 nm, between about 950 nm and about 1500 nm, between about 950 nm and about 1400 nm, or between about 950 nm and about 1300 nm, between about 1000 nm and about 1700 nm, between about 1000 nm and about 1600 nm, between about 1000 nm and about 1500 nm, between about 1000 nm and about 1400 nm, or between about 1000 nm and about 1300 nm. In certain embodiments, the absorbance spectrum of the composition is above about 900 nm.

[0096] The absorption spectrum of the composition has a peak wavelength between about 800 nm and 1400 nm, about 800 nm, about 900 nm, about 1000 nm, about 1100 nm, about 1200 nm, about 1300 nm, or about 1400 nm.

[0097] Absorbance spectra, such as plasmon resonance spectra, can be obtained using any suitable equipment and method, including but not limited to a visible near infrared spectrometer. The spectrum of the carrier may be subtracted from the spectrum of the total composition.

[0098] In certain embodiments, at least some of the clusters of metal nanoparticles have a coating layer that encapsulates the clusters. Multiple coating layers may be provided, for example, 2, 3, or 4 coating layers. The coating layer may include a polymer, a block copolymer, or a modified polymer. The coating layer may include an amphiphilic polymer. In certain embodiments, the coating layer is polyoxyethylene (40) stearate (e.g., "Myrj 52").

[0099] In certain embodiments, the coating layer has a thickness of about 0.5 nm to about 10 nm, hi certain embodiments, the coating layer has a thickness of less than about 1 nm.

[0100] The coating layer may comprise any suitable polymer, such as one or more of polyethylene glycol, polyvinyl chloride, poly-l-lysine, polylactic acid, poly(lactic-co-glycolic acid), polystyrene, and polyvinylpyrrolidone. The coating layer may comprise block copolymers, such as any member of the MYRJ™ and BRIJ™ families, including but not limited to polyoxyalkylenes with saturated or unsaturated alkyl chains (e.g., the BRIJ™ family); polyoxyethylene derivatives of saturated or unsaturated fatty acids, and / or high molecular weight polyoxyalkylene ethers with water-soluble, surface active wetting properties (e.g., the MYRJ™ family).

[0101] In certain embodiments, at least some of the clusters comprise metal nanoparticles functionalized with a stabilizing agent. In certain embodiments, the stabilizing agent comprises one or more of an amine, thiol, or carboxylic acid head group and a hydrophobic tail of any length and saturation, and optionally the stabilizing agent is oleylamine, octadecenethiol, oleic acid, or a combination thereof. In certain embodiments, the clusters of certain compositions comprise 9 nm gold particles capped with oleylamine.

[0102] In certain embodiments, a targeting agent is provided attached to the surface of the particle cluster. In certain embodiments where there is a coating on the particle cluster, the targeting agent is attached to the coating (e.g., an amphiphilic polymer). The targeting agent may include one or more of a small molecule ligand, a peptide, a polymer, a nucleic acid construct (including DNA and RNA aptamers), a protein, a nanobody, an affibody, a minibody, a diabody, or an antibody.

[0103] The targeting agent may bind to a marker of intravascular inflammation. The targeting agent may bind to one or more of P-selectin, E-selectin, and VE-cadherin. The targeting agent may be a ligand for P-selectin, E-selectin, or VE-cadherin, and may be a mixture of polymers, including, but not limited to, a mixture of fucose:sulfate (1:2), galactose:sulfate (1:2), or fucose:galactose:sulfate (1:1:1).

[0104] Carrier The carrier may comprise any suitable carrier. In certain embodiments, the carrier comprises an aqueous solution, a cream, or a gel. For biomedical applications, the aqueous carrier may comprise one or more of saline, water, or dextrose solution.

[0105] In certain other embodiments, the carrier comprises an organic solvent.

[0106] method With reference to FIG. 1, in certain aspects, a method for making an embodiment of the composition includes (i) reacting a metal nanoparticle precursor with a stabilizing agent to produce functionalized metal nanoparticles, (ii) dispersing the functionalized metal particles in a clustering agent to form metal particle clusters, and (iii) resuspending the metal particle clusters in a carrier to form the composition.

[0107] Any suitable metal nanoparticle precursors, stabilizing agents, clustering agents, and supports can be used to generate metal nanoparticle clusters with a variety of physical, optical, and chemical properties.

[0108] In certain embodiments, the metal nanoparticle precursor may include HAuCl4 or AgNO3, however, other metal salts are also possible metal precursors.

[0109] In certain embodiments, the stabilizing agent comprises one or more of an amine, thiol, or carboxylic acid head group and a hydrophobic tail of any length and degree of saturation. Optionally, the stabilizing agent is oleylamine, octadecene thiol, oleic acid, or a combination thereof.

[0110] In certain embodiments, the clustering agent is an organic solvent. Examples of clustering agents include, but are not limited to, butanol, ethanol, petroleum ether, butanol-hexane with or without pluronic F127, polyethylene oxide (40) stearate, and polyvinylpyrrolidone.

[0111] In certain embodiments, the reaction includes heating the metal nanoparticle precursor with a stabilizing agent. The heating can be carried out in any manner and to any suitable temperature for any suitable length of time to fully functionalize the metal particles with the stabilizing agent. The heating manner is not particularly limited. For example, the heating can be one or more of microwave heating, oven heating, oil bath heating, water bath heating, or mantle heating.

[0112] The method further includes, in certain embodiments, coating the metal particle cluster with a coating layer. The coating layer may include a polymer, such as an amphiphilic polymer. The polymer may include one or more of polyethylene glycol, polyvinyl chloride, poly-l-lysine, polylactic acid, poly(lactic-co-glycolic acid), polystyrene, and polyvinylpyrrolidone. The polymer may include modified polymers and / or block copolymers thereof. The block copolymers may include hydrophobic and hydrophilic domains (i.e., amphiphilic), such as, but not limited to, Pluronic family members, such as F127, MYRJ™, and / or BRIJ™ family members, such as polyethylene oxide (40) stearate, and polyvinylpyrrolidone. The coating step may be repeated to coat the particle cluster with multiple coatings.

[0113] In certain embodiments, the coating layer comprises a plurality of coating layers on at least one particle cluster. In certain embodiments, where the coating layer comprises an amphiphilic polymer, a plurality of amphiphilic coating layers are provided. It has been found that the addition of a polymer layer can increase the packing of the particle cluster.

[0114] In certain embodiments where the carrier in the composition is an aqueous carrier, the method further comprises separating the particle clusters from the clustering agent and suspending them in the aqueous carrier. Separation may be by one or more of centrifugation, sedimentation, size exclusion chromatography, or magnetic separation.

[0115] In certain embodiments, the method further comprises attaching a targeting agent to the surface of the particle cluster or to the coating layer (when present). The targeting agent may be any suitable agent, including but not limited to, a small molecule ligand, a peptide, a polymer, a nucleic acid construct (including DNA and RNA aptamers), a protein, a nanobody, an affibody, a minibody, a diabody, or an antibody. The targeting agent may bind to a marker of intravascular inflammation, such as P-selectin, E-selectin, or VE-cadherin. For example, but not limited to, the targeting agent may be a ligand of P-selectin, E-selectin, or VE-cadherin, such as a mixture of polymers, including a mixture of fucose:sulfate (e.g., 1:2), galactose:sulfate (e.g., 1:2), or fucose:galactose:sulfate (e.g., 1:1:1).

[0116] In certain embodiments, the method includes forming particle clusters having a given size by selecting an appropriate hydrophilicity of the clustering agent. More specifically, the size of the particle clusters can be increased by selecting a clustering agent with a higher hydrophilicity.

[0117] Uses of the compositions of the present technology may include, but are not limited to, as contrast agents for imaging and the like.

[0118] [Example] The present invention will be more readily understood by reference to the following examples, which are provided to illustrate the present invention and are not to be construed as limiting the scope of the present invention in any manner.

[0119] Unless otherwise defined or dictated otherwise by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0120] [Example 1] Gold-based particle clusters Referring to FIG. 2, a 40 mM solution (4 mL) of HAuCl4 in ethylene glycol (metal particle precursor) was added to 24.3 mM oleylamine (8 mL) (stabilizer) and 8 mL of ethylene glycol in a three-neck flask with stirring (950 RPM). Two of the three necks were capped with septa while the center neck was connected to a vacuum distillation adapter connected to a vacuumed and empty 5 mL flask. The three-neck flask was heated to 43° C. while applying vacuum and the solution was stirred for about 30 minutes until all bubbling had ceased. The flask was flushed with nitrogen gas and the vacuum adapter was removed. The solution was carefully poured (10 mL) into two microwave reactors which were flushed with nitrogen. The reactors underwent microwave synthesis in a chemical microwave (CEM Discover) (75 W power heating to 115° C., then held at this temperature for 90 seconds and cooled back to 50° C. upon discharging). The bath was decanted into a 50 mL Falcon tube containing a 15 g / L solution of polyethylene oxide stearate (Myrj 52) in n-butanol (coating layer). The solution was shaken overnight, centrifuged three times (1000×g, 10 min, RT), resuspended by sonication in butanol (clustering agent) in the first two centrifugations, and then finally resuspended by sonication in a 40 g / L solution of polyethylene oxide stearate (Myri 52) in ultrapure water (coating layer). The solution was again shaken overnight and centrifuged three times under the same conditions, resuspended in fresh ultrapure water after each centrifugation. The final solution was passed through a size exclusion chromatography column (SEC) to remove excess polymer. The final composition, containing particle clusters in suspension, was stored at 4° C.

[0121] Figure 3 shows the morphology and size distribution of particle clusters throughout their synthesis in butanol clustering without polymer coating (A), with a single polymer coating suspended in butanol (B), and with two polymer coatings in water (C). Size distributions were obtained through automated particle size analysis by ImageJ from two synthesis replicates of clusters, each with three regions on a transmission electron microscopy grid that were counted.

[0122] FIG. 4 shows the aqueous size and dispersibility of particle clusters fabricated with gold nanoparticles and double-coated with an amphiphilic polymer coating layer (AuSC@(Myrj 52)2). (A) Hydrodynamic size distribution of AuSC@(Myrj 52)2. (B) Zeta potential of AuSC@(Myrj 52)2. (C) Electrophoretic mobility of AuSC@(Myrj 52)2. All data were acquired from triplicate readings of AuSC@(Myrj 52)2 solutions in distilled water.

[0123] [Example 2] Adjusting the optical properties of the composition The optical properties of the particle clusters of Example 1 dispersed in butanol without polymer coating (AuSC bare) and with one or two polymer coatings of polyethylene oxide (40) stearate (AuSC@(Myrj 52) and AuSC@Myrj 52)2, respectively, were compared (Figure 5). The particle clusters without polymer coating showed a plasmon peak similar to the distinct peak known for single gold nanoparticles at around 9 nm (about 550 nm), but with a bathochromic shift from 550 to 700 nm (Figure 5A). A more prominent optical feature is the broad absorbance peak extending from 800 to 1400 nm. Both of these peaks persisted in butanol after single polymer coating of the particle clusters (Figures 5A and B, respectively). The NIR peaks became much sharper and more refined after further size focusing and aqueous transfer of the AuSC@(Myrj 52)2 spectrum when dispersed in water, but a large NIR broadband peak was retained between 1000 and 1400 nm (Figure 5C).

[0124] The formation of the large NIR peak was believed to be the result of large-scale plasmon hybridization throughout the 450 nm diameter particle cluster, resulting in a red-shifted plasmon band. Hybridization of such a large number of gold nanoparticles in one structure held in close proximity was the reason for the very large change in absorbance band compared to the plasmon hybridization red-shift reported in the previous literature. The unique optical properties of the double-coated gold particle cluster (AuSC@(Myrj 52)2) are believed to originate from the large number of individual gold nanoparticles that assemble together, entirely composed of close-packed gold nanoparticles from the core to the surface. This composition was confirmed through FIB-SEM imaging of the AuSC@(Myrj 52)2 clusters, where the superclusters were etched to reveal their core organization.

[0125] This example demonstrates how the optical properties of a composition can or cannot be tailored by coating particle clusters.

[0126] [Example 3] In silico simulation To better observe how large-scale hybridization is the result of the large NIR peak in the double-coated particle clusters of Example 2 (AuSc@(Myrj 52)2), these clusters were simulated in silico using a finite-difference time-domain (FDTD) model with the aim of reproducing particle clusters with similar optical properties as observed experimentally. These simulations were also used to determine the volume of the unit cells created by the particle clusters, since evaluation by X-ray diffraction did not produce any signal outside the unit cell for standard gold nanoparticles. Analogous to the molecular grain boundaries of gold nanoparticles, the particle clusters appear to assemble themselves in a unit cell-like manner. The unit cell volume is a measure that can aid in understanding how gold nanoparticle packing can affect the optical properties of particle clusters, as it ultimately governs the distance between particles. The simulated particle clusters were based on gold nanoparticles that were capped and oleylamine driven into the clusters using an amphiphilic solvent. The simulated particle clusters were assumed to be polymer coated with polyethylene oxide (40) stearate in butanol and then again in water, which likely resulted in a higher degree of polymer coating on the constituent gold nanoparticles nearer the surface than on the solvent-exposed surface, and more oleylamine on the particles closer to the cluster core. The heterogeneity of the surfaces of the constituent gold nanoparticles may result in a heterogeneous set of unit cells throughout the supercluster, with the core gold nanoparticles having smaller unit cells than those at the surface. When this gradient unit cell cluster was simulated with FDTD with two or three different unit cells, it produced absorbance spectra nearly identical to those observed experimentally. 14 nm 3 and the inner unit cell volume of 15 nm 3The outer side of the septum was used (Figure 6A) or 13 nm from the inner side to the outer side. 3 , 14nm 3 , and 15 nm 3 Two different simulations using (Figure 6B) yielded very similar absorbance spectra, especially in comparison to the experimental data. Clusters of uniform unit cell volume did not resemble the experimental data (Figure 7). Both the absorption and scattering components of the photophysical behavior of the simulated particle clusters were extracted from the extinction data, demonstrating that scattering is the dominant interaction in the visible to NIR wavelengths, but there is still a high degree of light absorption in the visible range. Importantly, scattering is the dominant mode of light interaction in the NIR-II. This dual mode of light interaction (i.e., both absorption and scattering) spans the visible-NIR-NIR-II spectrum, making these clusters interesting materials for widespread use. Figure 8 shows finite-difference time-domain simulation data for AuSC@(Myrj 52)2 superclusters using various unit cells. Each curve represents the unit cell volume range (inside to outside) of the simulated particle clusters and the resulting extinction (combined absorption and scattering) spectrum. The simulation is in an aqueous environment that includes a 10 nm polymer (Myrj 52) coating around the particle clusters.

[0127] Figure 9 shows finite-difference time-domain simulation data of particle clusters with a double polymer layer (AuSC@(Myrj 52)2) using various polymer coating thicknesses. The clusters were 15 nm 2 particles composed of 9 nm diameter gold nanoparticles. 3 The unit cell was simulated by finite-difference time-domain measurements. The surrounding simulated environment was water. The thickness of the simulated polymer coating, up to a thickness of 50 nm, did not change the optical properties of the particle clusters.

[0128] [Example 4] Tuning the optical properties of the composition using different stabilizing agents and different clustering agents Table 1 shows various synthesis conditions for forming gold particle clusters, including reaction concentrations and conditions, post-processing steps, and the resulting absorbance peaks and appearance of the superclusters. As can be seen, the optical properties of the composition can be tailored by adapting the reagents used to make the composition.

[0129] [Table 1] JPEG2025508320000003.jpg255153JPEG2025508320000004.jpg255154JPEG2025508320000005.jpg25591

[0130] [Example 5] Compositions for use as IV-OCT Intravascular optical coherence tomography (IV-OCT) is commonly used in interventional cardiology evaluation to image cardiovascular health and guide stent placement. However, IV-OCT is limited to anatomical imaging due to the current lack of contrast agents (agents that can provide specific signal enhancement). IV-OCT relies on backscattered incoherent NIR-II light (center wavelength around 1300 nm), which is well suited for contrast enhancement by gold particle clusters. The composition of Example 1 was prepared, which included gold double-coated particle clusters (AuSC@(Myrj 52)2) in a saline carrier. The strong ionic solvent did not affect the structure or optical properties of the particle clusters (Figure 10). Two-dimensional (2D) IV-OCT scans of various concentrations of particle clusters suspended in glass pipettes were performed to evaluate the contrast enhancement effect (Figure 11). A saline-soluble commercially available gold nanoparticle solution (mVivo, Medilumine Inc.) was used as a reference sample. The IV-OCT signal enhancement generated by the composition was significantly greater (>10-fold) than that generated by the reference gold nanoparticles, even after normalization to the total gold content of the solution (FIG. 11). Even microgram quantities of AuSC@(Myrj 52)2 resulted in a 3-fold signal enhancement. Large individual 500 nm gold nanoparticles (not superclusters) were also evaluated and did not show a strong signal compared to the composition's AuSC@(Myrj 52)2 clusters (approximately 1.8-fold signal enhancement for superclusters compared to AuNPs, normalized to the number of particles in solution) (FIG. 12).

[0131] A vascular phantom was prepared using particle clusters of the metal and gold nanoparticles of the present technology in suspension in agarose (FIG. 13) to evaluate the dynamic implementation of the more commonly used IV-OCT, producing longitudinal images by pulling the imaging catheter back through the vasculature (FIG. 13). Signal intensity over the scan distance was mapped (FIG. 13), and only the particle clusters of the composition provided contrast enhancement above background. The particle clusters also highlighted a significant amount of detail in the agarose, such as air pockets and fractures that are not easily discernible in the absence of contrast agent.

[0132] The composition containing AuSC@(Myrj 52)2 particle clusters was also applied in in vivo imaging of the abdominal aorta (AA) of Sprague-Dawley rats. The AA was imaged while flushed with saline (Figure 14) and then imaged while flushed with a 0.5 mg / mL AuSC@(Myrj 52)2 solution in saline. There is a clear contrast to the flushed space that is distinguishable from the signal created from the walls of the AA. We then flushed the AA with additional saline to demonstrate that the particles can be easily washed out of the field of view after imaging (Figure 14).

[0133] By addition of targeting groups at the surface of particle clusters specific for markers of inflammation, this difference in contrast before and after flushing could be used to detect intravascular inflammation before major morphological changes occur (Figure 15A-I). The polymer coating could be easily functionalized with targeting groups capable of binding to the biomolecular target of interest (i.e., P-selectin, E-selectin, VE-cadherin, etc.), all markers of intravascular inflammation, allowing molecular imaging by IV-OCT (Figure 15A). The targeting agent could be a small molecule ligand, peptide, aptamer, or antibody conjugated to the polymer coating using well-established mechanisms (e.g., lbrich K et al., Chem Rev, 2016;116(9):5338-5431, the contents of which are incorporated herein by reference).

[0134] Figure 15B shows transmission electron micrographs of AuSCs functionalized with different combinations of targeting ligands as indicated (left) and the degree of AuSC binding to P-selectin in vitro for different formulations of AuSc targeting (right). Figure 15C shows intravascular optical coherence tomography of Sprague-Dawley rat abdominal aorta after induction of intra-arterial inflammation, before and after introduction of non-targeted (left) or targeted AuSCs (middle and right), demonstrating successful targeting of AuSc functionalized with targeting ligands.

[0135] Although the present invention has been described in detail with reference to embodiments thereof, these embodiments are provided to illustrate, not limit, the invention. Other embodiments may be made which employ the principles of the invention and are encompassed within its spirit and scope as defined by the claims appended hereto.

[0136] The contents of all documents and references cited herein are hereby incorporated by reference in their entirety.

Claims

1. 1. A composition comprising a plurality of particle clusters in a carrier, the particle cluster comprises a plurality of metal nanoparticles functionalized with a stabilizer, the particle cluster having a coating layer; the stabilizing agent comprises one or more of an amine, a thiol, or a carboxylic acid head group and a hydrophobic tail; the coating layer comprises an amphiphilic polymer; the metal nanoparticles comprise a plasmonic metal, a plasmonic metal alloy, or a plasmonic metal oxide; The carrier comprises an aqueous solution or a polar organic solvent. composition.

2. The metal nanoparticles are silver or gold particles having a size range of about 1 nm to about 500 nm, or a size range of about 1 nm to about 100 nm. The composition of claim 1.

3. The stabilizer is oleylamine, octadecene, oleic acid, or a combination thereof. The composition of claim 1.

4. the amphiphilic polymer comprises one or more of polyethylene glycol, polyvinyl chloride, poly-l-lysine, polylactic acid, poly(lactic-co-glycolic acid), polystyrene, polyvinylpyrrolidone, or modified polymers or block copolymers thereof; The composition of claim 1.

5. The amphiphilic polymers include polyoxyalkylenes with saturated or unsaturated alkyl chains (e.g., the BRIJ™ family), polyoxyethylene derivatives of saturated or unsaturated fatty acids, and / or high molecular weight polyoxyalkylene ethers that are water-soluble and have surface-active and wetting properties (e.g., the MYRJ™ family). The composition of claim 1.

6. further comprising a targeting agent attached to the surface or coating of the particle cluster; the targeting agent comprises one or more of a small molecule ligand, a peptide, a polymer, a nucleic acid construct (including DNA and RNA aptamers), a protein, a nanobody, an affibody, a minibody, a diabody, or an antibody; The composition of claim 1.

7. the targeting agent binds to a marker of intravascular inflammation; The composition of claim 6.

8. the targeting agent binds to one or more of P-selectin, E-selectin, and VE-cadherin; The composition of claim 6.

9. the targeting agent comprises a mixture of polymers; The mixture comprises a mixture ratio of fucose:sulfate (1:2), galactose:sulfate (1:2), or fucose:galactose:sulfate (1:1:1), The composition of claim 6.

10. the carrier comprises saline, water, or 5% dextrose in water; The composition of claim 1.

11. the particle clusters have an average diameter of about 250 nm to about 1500 nm, or about 300 nm to about 500 nm; The composition of claim 1.

12. the metal nanoparticles are gold nanoparticles, the stabilizer is oleylamine, the coating layer comprises Myrj™ 52; the carrier is water; The composition of claim 1.

13. 10. A method for producing a composition as defined in claim 1, comprising: (i) providing a solution comprising a metal nanoparticle precursor and a stabilizing agent; (ii) reacting the metal nanoparticle precursor with the stabilizing agent to form functionalized metal nanoparticles; (iii) suspending the functionalized metal nanoparticles in a solution containing a coating agent and a clustering agent to obtain coated metal particle clusters; (iv) suspending the coated metal particle clusters in a carrier to obtain the composition; Including, the stabilizing agent comprises one or more of an amine, a thiol, or a carboxylic acid head group and a hydrophobic tail; the coating layer comprises an amphiphilic polymer; the metal nanoparticle precursor comprises a plasmonic metal precursor, a plasmonic metal alloy precursor, or a plasmonic metal oxide precursor; the carrier comprises an aqueous solution or a polar organic solvent; the clustering agent comprises an organic solvent; method.

14. The metal nanoparticle precursor is a gold particle precursor or a silver particle precursor. The method of claim 13.

15. The metal nanoparticle precursor is HAuCl ４ or AgNO ３ That is, 15. The method of claim 14.

16. the amphiphilic polymer comprises one or more of polyethylene glycol, polyvinyl chloride, poly-l-lysine, polylactic acid, poly(lactic-co-glycolic acid), polystyrene, polyvinylpyrrolidone, or modified polymers or block copolymers thereof; The method of claim 13.

17. the reacting step includes heating a metal nanoparticle precursor with a stabilizing agent; The heating is one or more of microwave heating, oven heating, oil bath heating, water bath heating, or mantle heating; The method of claim 13.

18. The stabilizer is oleylamine, octadecene, oleic acid, or any combination thereof. The method of claim 13.

19. the clustering agent is one or more of butanol, ethanol, petroleum ether, and butanol-hexane; The method of claim 13.

20. the carrier in the composition is an aqueous solution; the method further comprising the steps of separating the particle clusters from the clustering agent and suspending the separated particle clusters in an aqueous solution; the separation is carried out by one or more of centrifugation, sedimentation, size exclusion chromatography or magnetic separation; The method of claim 13.

21. The metal nanoparticle precursor is HAuCl ４ and the stabilizer is oleylamine, the coating comprises Myrj™ 52; the clustering agent is n-butanol; the carrier is water; The method of claim 13.

22. 10. Use of the composition of claim 1 as a contrast agent.

23. 10. Use of the composition of claim 1 in biomedical imaging.

24. 10. A contrast agent for biomedical imaging comprising the composition of claim 1.

Images